Study On Genetic Screening Of Idiopathic Pulmonary Fibrosis And Its Acute Exacerbating Mechanism

Background:The association between a single nucleotide polymorphism(SNP)rs35705950 in the Mucin 5B(MUC5B)and idiopathic pulmonary fibrosis(IPF)has been studied in different racial populations and is notably in Caucasian populations.The association between rs35705950 and IPF is presented in Japanese population but not in Korean population.Another SNP rs2034650 in the Isovaleryl-CoA dehydrogenase(IVD)gene is reported to be associated with IPF in Korean population.This study was designed to confirm the association between these two SNP loci and IPF in a Chinese Han populationShort telomeres are risk factors and associated with poor survival for idiopathic pulmonary fibrosis(IPF).In this study,we aim to examine the relationship between telomere length and the pathogenesis of IPF by examining the telomere length of UIP and possible UIP pattern according to HRCT and healthy controls group,combined with clinical data analysis.Meanwhile,as a basis for further study of the relationship between the accuracy of telomere length and the causal relationship between IPF pathogenesis.Acute exacerbation of idiopathic pulmonary fibrosis(AE-IPF)has high short-term mortality with unknown causes.It is challenging to predict that acute malignancy in this acute exacerbation of IPF is clinically present.In this part of the study,our aim was to find out whether there were different miRNAs expressing between AE-IPF and stable IPF,which could be used as reliable biomarkers to predict whether IPF would develop AE-IPFMethods:Genomic DNA was extracted from blood samples in 132 IPF patients and 136 healthy controls,and rs35705950 and rs2034650 were detected by commercially available genotyping assay.Selected samples with different genotypes were confirmed by Sanger sequencing.Leukocyte TLs were measured by quantitative polymerase chain reaction(q-PCR)in patients with suspected IPF upon HRCT at the time of the initial enrolment assessment in Nanjing Drum Tower Hospital Affiliated to Medical School of Nanjing University.Three pulmonary specialists evaluated HRCT and each case was classified into UIP and possible UIP according to ATS/ERS guideline.For patients with UIP pattern,three pulmonary specialists scored HRCT findings including the extent of ground glass opacities(GGO),reticulations,honeycombing and fibrosis score independently.Univariate analysis and multivariate linear regression models were used to identify factors associated with TL in patients with UIP pattern.The human fibrosis-related miRNAs were designed to detect the expression of miRNAs in plasma of three AE-IPF patients,three stable IPF(S-IPF)patients and three normal controls(NC).Based on the results of the chip analysis,miRNAs were selected for the differential expression of miRNAs between AE-IPF patients and plasma of S-IPF patients.Large sample fluorescence quantitative Q-PCR validation studies were performed in 12 AE-IPF patients,45 S-IPF patients and 51 healthy control subjects in plasma.The diagnostic accuracy of miRNAs with differentially expressed differences was analyzed using the receiver operating characteristic curve(ROC).DIANA-miRPath predicts that miRNAs are verified to interact with signaling pathways and cellular processes.Results:The genotype distributions of rs35705950 in Chinese IPF patients were significantly different from those in healthy subjects(P<0.05).The rs35705950 T allele frequencies were 3.8%in IPF cohort and 0.7%in control population,and the T allele frequency was significantly higher in IPF patients than in healthy controls(P<0.05).The genotype distributions and the G allele frequencies of rs2034650 were not significantly different between this Chinese IPF cohort and control population.A total of 130 patients including 108 with UIP pattern and 22 with possible UIP pattern in HRCT were enrolled in this study according to the exclusive criteria.TLs in patients with UIP pattern were significantly shorter(1.28±0.920)compared to those with possible UIP patterns(1.77±0.961,P=0.027)and healthy controls(3.06±1.31,P=0.000).After adjusting for sex and age,univariate analysis showed that extent of reticular and honeycombing,fibrosis scores and pulmonary function data including FVC%and DLCO%predicted were associated with TL.In multivariate linear regression models,the extent of honeycombing was the only factor independently associated with TL.According to the results of the chip analysis,6 miRNAs were differentially expressed between AE-IPF and S-IPF patients(P<0.05).In the validation study,let-7d-5p was reduced in S-IPF compared to healthy controls and further decreased in AE-IPF((0.0007±0.0005vs0.003±0.002,P<0.01 and 0.0003±0.0002 vs 0.003±0.002,P<0.01),but significantly increased in AE-IPF(0.0023±0.002vs0.0003±0.0003,P<0.01),,and the expression of miR-25-3p was significantly decreased in S-IPF patients(0.0002±0.0001vs0.0003±0.002,P<0.01).The AUCs of miR-25-3p and let-7d-5p under the curve were 0.83 and 0.75,respectively,which were greater than the threshold of 0.7 for the diagnostic study in the receiver operating characteristic curve(ROC)analysis.When the sensitivity of immobilization was 90%,the AUC improved from 50%to 66.7%after two miRNA combinations.Functional prediction of miRNAs suggests that the loss of anti-fibrosis capacity and the availability of uncontrolled cell growth may be required in the pathogenesis of AE-IPF.In summary,miR-25-3p and let-7d-5p in plasma were differentially expressed between AE-IPF and S-IPF.These two miRNAs may be a potential biomarker for the diagnosis of AE-IPF from IPF.Conclusions:The SNP rs2034650,detected in Chinese population for the first time,was not associated with IPF in this Chinese Han cohort.However,the association between rs35705950 and IPF,similar to but not as strong as in Caucasian populations,was also presented in Chinese Han population.The results showed that telomere lengths in circulating leukocytes were associated with the extent of honeycombing in IPF,which provided evidence for telomere shortening involving in the progressive pathogenesis of IPF.Further work,particularly longitudinal studies will be needed to add precision that TL is causally related to IPF pathogenesis.MiR-25-3p and let-7d-5p in plasma were differentially expressed between AE-IPF and S-IPF.The combined analysis of these two miRNAs may be a potential biomarker for predicting AE-IPF from IPF.